【作者】 肖春桥；

【导师】 池汝安；

【作者基本信息】 中南大学 ， 生物工程， 2009， 博士

【摘要】 我国磷矿资源丰富,但其中大部分是中低品位磷矿,这些中低品位磷矿在我国目前对磷矿资源的开发利用方面基本上是处于被废弃的地位,造成了资源的极大浪费。本文以微生物溶解中低品位磷矿为主线,分别从异养菌溶磷和自养菌溶磷两个方面展开研究,以期为解决我国丰富的中低品位磷矿资源利用难题提供理论依据。在异养菌溶磷研究部分,本文首先研究了溶磷异养菌的分离筛选及其对中低品位磷矿粉的溶解作用。首次从湖北省境内磷矿生态环境中分离筛选出一株溶磷细菌YC和三株溶磷真菌HB1、HB2和HB3。通过对四株分离菌株进行形态、生理生化及分子鉴定,结果表明,YC为嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia,S.maltophilia）,HB1为扩展青霉（Penicillium expansum,P.expansum）、HB2为分枝毛霉（Mucor ramosissimus,M.ramosissimus）,HB3为克鲁斯假丝酵母（Candida krissii,C.krissii）。本文是首次报道这些菌株为溶磷微生物。探讨了四株分离菌株的溶磷机理。结果表明,它们在代谢过程中分泌的有机酸如葡糖酸、柠檬酸和草酸等对磷酸三钙以及磷矿粉的溶解起到了关键作用。另外,四株分离菌株的溶磷能力与它们在培养液中的数量呈正相关,与培养液pH呈负相关。研究了四株分离菌株溶解磷矿粉的最佳工艺条件。S.maltophiliaYC溶解磷矿粉的最佳工艺条件为:培养温度,29～32℃;初始pH,6.5～7.0;磷矿粉浓度,2.0g/L;磷矿粉粒度,0.04mm;振荡速率,140r/min;菌液接种量,25%。三株溶磷真菌（P.expansum HB1、M.ramosissimus HB2和C.krissii HB3）分别在培养温度为32℃、振荡速率为160r/min以及磷矿粉浓度和粒度分别为2.5g/L和0.04mm时,溶磷量达到最大。另外,在溶磷过程中,尽量减少有毒离子如Ag⁺的存在,或者加入一些螯合剂如EDTA等,有助于磷矿粉中可溶性磷的释放。以盆栽小麦为研究对象,探讨了接种三株溶磷真菌对小麦幼苗生长及磷素代谢的影响。结果表明,三株溶磷真菌能通过溶解土壤中的磷矿粉从而提高土壤有效磷含量,并且有显著促进小麦幼苗茎和根长、增加茎和根的干重,以及促进小麦幼苗对磷、氮等营养元素吸收的作用。在自养菌溶磷研究部分,本文首先研究了利用嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans,At.f）和嗜酸氧化硫硫杆菌（Acidithiobacillus thiooxidans,At.t）分别在黄铁矿粉和硫磺粉存在下对中低品位磷矿粉的溶解作用。结果表明,At.f和At.t能分别通过氧化黄铁矿粉和硫磺粉产生硫酸,形成有利于它们生长的酸性环境,同时产生的硫酸可以有效溶解磷矿粉,浸出其中的磷。在pH均为2.5的条件下,利用At.f和At.t溶解磷矿粉比稀硫酸直接溶解更为有效,其对磷的浸出率远高于稀硫酸直接溶解时的浸出率。探讨了At.f和At.t分别在黄铁矿粉和硫磺粉存在下溶解磷矿粉的最佳工艺条件。结果表明,在黄铁矿粉存在下,At.f溶解磷矿粉浸出其中磷的最佳工艺条件为:培养温度,30℃;初始pH,2.0;振荡速率,140r/min;磷矿粉浓度,5g/L;At.f接种量,25%;黄铁矿粉加入量,25g/L;HPO₄^2-浓度,1.0g/L;NH₄⁺浓度,1.5g/L;Mg²⁺浓度,0.5g/L。在硫磺粉存在下,At.t溶解磷矿粉浸出其中磷的最佳工艺条件为:At.t接种量,25%;磷矿粉浓度,5g/L;初始pH,2.0;硫磺粉加入量,10g/L。为了促进At.f和At.t对磷矿粉中磷的浸出,应尽量将磷矿粉和黄铁矿粉磨细,减少培养液中Fe³⁺的累积,同时尽量使用驯化菌作为接种菌。另外,加入一些表面活性剂如吐温80等也能促进At.f和At.t对磷的浸出。研究了At.f的诱变和固定化处理及其对磷矿粉的溶解作用。结果表明,采用诱变和固定化处理能增强At.f的氧化活性,促进其对磷矿粉中磷的浸出。诱变处理对黄铁矿粉存在下At.f浸出磷矿粉中磷的最佳工艺条件是:15W紫外灯下15cm照射At.f5min;800W微波辐射At.f40 sec;浓度为1.0%的硫酸二乙酯诱变处理15min。以海藻酸钠、聚乙烯醇和卡拉胶固定处理At.f,结果发现其中以海藻酸钠固定At.f时的溶磷效果最好,培养第5d,其At.f氧化活性是游离At.f氧化活性的3.5倍,其对磷矿粉中磷的浸出率是游离At.f浸出率的1.28倍。更多还原

【Abstract】 There are abundant of rock phosphates（RPs） in China,but most of them are middle-low grade and often abandoned today.Heterotrophic and autotrophic microorganisms were used to solubilize middle-low grade RP, respectively,in this study.It is expected that the results of this study will provide a theoretical basis to solve the utilization problems of middle-low grade RPs in China.The isolation and screening of heterotrophic phosphate solubilizing microorganisms and phosphate solubilization by these microorganisms were studied in this study.A bacterial strain YC and three fungal strains of HB1,HB2 and HB3 were isolated from phosphate mines in Hubei province（PR China）.They are identified as Stenotrophomonas maltophilia（S.maltophilia）,Penicillium expansum（P.expansum）,Mucor ramosissimus（M ramosissimus） and Candida krissii（C.krissii）, respectively.It is the first time to report these strains as phosphate solubilizing microorganisms in this study.The mechanisms of phosphate solubilization by the isolates were discussed.Results show that organic acids including gluconic acid,citric acid and oxalic acid,which were released by the isolates,played key roles on the solubilization of tricalcium phosphate and RP.Moreover,there have a positive correlation between concentration of soluble phosphorus and population of the isolates,and a negative correlation between concentration of soluble phosphorus and pH in the culture medium.The optimum conditions for the isolates to solubilize middle-low grade RP were studied.The optimum conditions to release soluble phosphorus from RP by S.maltophilia YC are:culture temperature, 29-32℃;initial pH,6.5-7.0;RP concentration,2.0 g/L;RP particle size, 0.04 mm;shaking speed,140 r/min;and inoculation of S.maltophilia YC, 25%.The maximal contents of soluble phosphorus released by three fungal isolates（P.expansum HB1,M.ramosissimus HB2 and C.krissii HB3） are attained when cultured at 32℃,shaked at 160 r/min,and added with RP concentration and particle size at 2.5 g/L and 0.04 mm, respectively.In addition,it will facilitate the solubilization of RP by the isolates to minimize the existence of toxic ions such as silver ion,or add some chelating agents such as EDTA,etc.Wheat seedling growth promoting experiments by three fungal isolates under pot culture conditions were investigated in this study. Results show that all the fungal isolates promoted the length and dry weight of the shoot and root,and phosphorus and nitrogen uptake of wheat seedlings in field soil containing RP,thus demonstrating the capability of the isolates to convert insoluble forms of phosphorus into plant available forms from RP,and therefore hold great potential for development as biofertilizers to enhance soil fertility and promote plant growth.Autotrophic microorganisms,Acidithiobacillus ferrooxidans（At.f） and Acidithiobacillus thiooxidans（At.t） were used to bioleach soluble phosphorus from middle-low grade RP containing pyrite and sulfur, respectively,in this study.Sulfuric acid was produced by the bio-oxidation of pyrite and sulfur by At.f and At.t,respectively.The production of sulfuric acid not only benefits the growth of At.f and At.t, but also solubilizes RP.Under the same pH condition（pH is 2.5）,the bioleaching of soluble phosphorus from RP by At.f and At.t are more effective than sulfuric acid leaching.The optimum conditions for At.f and At.t to bioleach soluble phosphorus from RP were studied.The optimal technological parameters of bioleaching soluble phosphorus from RP by At.f are:culture temperature,30℃;initial pH,2.0;shaking speed,140 r/min;RP concentration,5 g/L;inoculation of At.f,25%;pyrite addition,25 g/L; HPO₄^2- concentration,1.0 g/L;NH₄⁺ concentration,1.5 g/L;Mg²⁺ concentration,0.5 g/L.The optimal technological parameters of bioleaching soluble phosphorus from RP by At.t are:inoculation of At.t, 25%;RP concentration,5 g/L;initial pH,2.0;sulfur addition,25 g/L.In order to promote the bioleaching of soluble phosphorus,RP and pyrite should be ground as fine as possible;the accumulation of Fe³⁺ in the culture medium should be minimized,and used adapted bacteria as inoculants.In addition,the addition of surfactants such as Tween 80 will promote the bioleaching of soluble phosphorus from RP.The bioleaching of soluble phosphorus from RP by mutagenic and immobilized At.f were studied.Results show that the treatments of At.f by mutagenesis and immobilization could enhance the oxidative activity of At.f,and increase the leaching rate of soluble phosphorus.The optimal technological parameters of bioleaching soluble phosphorus from RP by mutagenic At.f are:irradiating with 15 W ultraviolet ray lamp for 5 min at a distance of 15 cm;radiating with 800 W microwaves for 40 sec; mixing with 1.0%diethyl sulfate for 15 min.The immobilized At.f by sodium alginate has the biggest leaching rate of soluble phosphorus among the immobilized At.f by sodium alginate,polyvinyl alcohol and carrageenan.The oxidative activity and leaching rate of soluble phosphorus of immobilized At.f by sodium alginate are of 3.5 and 1.28 times than that of bioleaching by free At.f,respectively.更多还原